ABSTRACT Interspecific hybridization is a useful tool in genetic
improvement of agriculture and aquaculture species. The Pacific oyster
(Crassostrea gigas) and the Hong Kong oyster (Crassostrea hongkongensis)
are both important aquaculture species in China. To determine whether
these 2 species can hybridize and produce viable offspring, we conducted
2 x 2 factorial crosses between them. Asymmetry in fertilization was
observed when C. hongkongensis eggs were fertilized readily by C. gigas
sperm, but the reciprocal cross resulted in no fertilization. Embryos
from C. hongkongensis female x C. gigas male crosses developed normally
without noticeable defects, although their survival rate to D-stage was
less than embryos of the two intraspecific crosses. From D-stage to
metamorphosis, larvae of hybrid crosses had slower growth and a lower
survival rate than that of intraspecific crosses. Nevertheless, 0.57% of
hybrid D-larvae survived to spat stage at day 90. Hybrid spat had good
survival (78.9%) to 1 y of age, but were significantly (P < 0.001)
smaller than oysters of intraspecific crosses. Gonadal development was
absent or retarded in most hybrids at 1 y of age, although some hybrids
(39.2%) produced mature gametes. Our results show that hybridization
between C. gigas and C. hongkongensis is possible in one direction. Some
hybrids are viable, partly fertile, and can be used potentially for gene
introgression between these two species.

Interspecific hybridization is a useful tool in genetic improvement
of agriculture and aquaculture species (Briggs & Knowles 1967,
Hulata 1995, Bartley et al. 2001). Hybrids may be used directly as stock
for farming or they can be used for gene introgression between species.
Studies on interspecific hybridization may also provide insight on
mechanisms of prezygote and postzygote barriers to hybridization, which
are important to our understanding of speciation and evolution (Pahimbi
1992, Palumbi 1994).

Oysters are important aquaculture species, and there is
considerable need for genetic improvement of oysters because most
cultured stocks are undomesticated and suffer from problems of slow
growth, disease, and summer mortality (Guo 2009). Some genetic
improvement of oyster stock has been achieved through selective breeding
and chromosome set manipulation (Boudry 2009, Guo et al. 2009).
Hybridization as a tool for oyster breeding has also received some
attention. There has been strong interest in hybridizing Crassostrea
virginica and Crassostrea gigas, because the latter is resistant to two
lethal diseases of the former species (Calvo et al. 1999).
Unfortunately, despite repeated attempts, C. virginica and C. gigas
cannot be hybridized because of postzygotic barriers (Allen et al.
1993).

Many attempts at hybridization have been reported in Crassostrea
species, although in most early studies the claims of hybridization were
not supported by genetic confirmation of hybrids (Gaffney & Allen
1993). Sperm and larval contamination is a common occurrence in oyster
spawning and larval culture. Claims of hybridization without genetic
confirmation must be viewed with caution. The production of viable
hybrids has been confirmed with genetic analysis in the following
crosses: C. gigas x Crassostrea angulata (Soletchnik et al. 2002, Huvet
et al. 2004), C. gigas x Crassostrea rivularis (Allen & Gaffney
1993, Que & Allen 2002), C. gigas x Crassostrea sikamea (Banks et
al. 1994, Camara et al. 2008), and Crassostrea ariakensis x C. sikamea
(Xu et al. 2009). Asymmetry in fertilization success has been reported
in several crosses, providing opportunities for studying the evolution
of the proteins involved in sperm-egg interaction.

There are at least 5 Crassostrea oyster species that occur
naturally along the coast of China: C. gigas, C. ariakensis, C. sikamea,
C. angulata, and Crassostrea hongkongensis (Wang et al. 2006, Guo et al.
2008). Often, some of these species overlap in distribution. In North
China, C. ariakensis coexists with C. gigas. In central and southern
China, C. ariakensis, C. hongkongensis, C. angulata, and C. sikamea may
be found in the same estuary in various combinations. Studying
hybridization among these sympatric species may contribute to our
understanding of mechanisms of reproductive isolation and speciation.

Although C. gigas and C. hongkongensis do not overlap in
distribution, both are major aquaculture species in China. The oyster C.
gigas is traditionally cultured in North China, but it has been
introduced to southern China for aquaculture production (Guo et al.
1999, Guo 2009). The oyster C. hongkongensis is the most important
oyster species cultured in southern China (Wang et al. 2004, Guo et al.
2006). It is a large oyster species and commands high market prices. It
is found in low-salinity estuaries from the Fujian to Guangxi provinces,
with populations centered in the Guangdong province. Although C.
hongkongensis thrives in the warm and low-salinity waters of southern
China, C. gigas is adapted to the temperate and high-salinity waters of
northern China. These local adaptations may have given them different
abilities or unique physiological traits in environmental tolerance that
may be useful for selective breeding. Tolerance of heat stress in C.
hongkongensis, for example, would be a useful trait for C. gigas, which
often suffers from summer mortality. The tolerance of high salinity of
C. gigas, if transferred successfully may make C. hongkongensis grow
better in high-salinity waters. Thus, hybridization between C. gigas and
C. hongkongensis is potentially useful in the genetic improvement of
these two important aquaculture species. However, no hybridization
between these two species has been reported, because C. hongkongensis is
a newly described species (Lain & Morton 2003, Wang et al. 2004).

To determine whether hybridization between C. gigas and C.
hongkongensis is possible, we conducted 2 x 2 crosses between the two
species. Here we report that hybridization between the two species is
possible in one direction. Despite low survival, slow growth, and
retarded gonadal development, some hybrids can reach sexual maturation
and produce functional gametes, raising the possibility of gene
introgression between these two species.

MATERIALS AND METHODS

Oysters, Gamete Collection, and Hybridization Crosses

The oyster C. hongkongensis was collected from Shenzhen (Guangdong,
China), and C. gigas were collected from Yantai (Shandong, China).
Oysters of both species were about 2 y old. They were transported to the
hatchery of Qingdao Laodong Aquaculture Breeding Company for
conditioning in February 2009. They reached sexual maturity in early
April, and our experiments were conducted from mid April to mid May
2009.

Gametes from the two species were collected by dissecting gonads.
Before gamete collection, the two species were identified by shell
morphology and differences in gill tube structure as described in Wang
et al. (2004). After gamete collection, a piece of adductor muscle from
each parental oyster used for hybridization was fixed in 95% ethanol for
subsequent confirmation with genetic markers (described later).

Egg suspension was passed through an 80-[micro]m nylon screen to
remove large tissue debris, and eggs were caught and washed on a
20-[micro]m screen. For each species, eggs from 3 females were pooled
and then divided equally into two 2-L beakers. Eggs were examined under
a microscope to ensure no uncontrolled fertilization had occurred, as
indicated by the absence of polar bodies. After confirming no
uncontrolled fertilization, eggs in the 2 beakers were fertilized with
pooled sperm from 3 C. gigas and 3 C. hongkongensis males, respectively.
Thus, a 2 x 2 factorial cross was created producing 4 groups: C. gigas
[female] x C. gigas [male] (GG), C. gigas [female] x C. hongkongensis
[male] (GH), C. hongkongensis [female] x C. gigas [male] (HG), and C.
hongkongensis [female] x C. hongkongensis [male] (HH). Sperm were added
within 60 min after gamete collection to a density of about 20-25 sperm
surrounding an egg in intraspecific crosses. For hybrid crosses, about
50% more sperm were added. Fertilization was conducted at 25[degrees]C
in filtered seawater with a salinity of 30. The experiment was
replicated 9 times using 9 sets of parents.

Larval Rearing

Fertilized eggs were sampled and held in beakers to evaluate
fertilization success. The remaining eggs from each group were counted
and cultured in a 60-L bucket for incubation at a density of 30-40
eggs/mL. At 27 h after fertilization, D-larvae from each group were
collected on a 40-[micro]m screen and reared in a 60-L bucket at 5
larvae/mE Larvae were fed with Isochrysis galbana on days 0-6, and a
mixture of Platymonas subcordiformis and I. galbana (1:1) after day 6.
Feeding was increased gradually from 6,000-80,000 cells/mL/day. Seawater
was changed completely every 3 days. The culture seawater was maintained
at 24.8 26.0[degrees]C.

Settlement, Nursery, and Grow-out

When most larvae developed an eye spot and foot, strings of
corrugated plastic plates were placed in buckets as cultch. Larvae set
within 8 days, and newly settled spat were nursed in buckets for 2 wk to
prevent contamination from wild spat. Subsequently, all spat were
transported to concrete tanks (8.0 x 5.0 x 1.2 m) and fed with Chlorella
vulgaris at 80,000-10,000 cells/ mL/day. Water was changed 30% once
daily. At day 60, spat were detached, transferred into spat bags, and
hung on suspended longlines in large shrimp ponds. Spat bags were
changed periodically from small to large mesh sizes. During the grow-out
period from July 2009 to June 2010, water temperature ranged from
6.0-30.2[degrees]C and salinity ranged from 24-30.

Sampling, Measurements, and Data Analysis

Sixty minutes after fertilization, a 2-mL sample was collected from
each group, and the number of fertilized (as indicated by polar bodies
or cell division) and unfertilized eggs were counted. Fertilization
level was calculated as the percentage of fertilized eggs to the total
number of eggs. Egg diameter was determined by measuring 90 eggs under a
microscope fitted with a calibrated eyepiece micrometer. Larval size was
measured the same way for 30 larvae per group at D-stage and every 3
days thereafter. At 27 h postfertilization, the number of D-stage larvae
was determined for each group, and percent survival of fertilized eggs
to D-stage was determined. Subsequently, larval survival was determined
as a percentage of D-stage larvae surviving to different days
postfertilization.

During nursery and grow-out, 30 spat or oysters were sampled
randomly and their shell height was measured with an electronic Vernier
caliper at days 90, 180, and 360. Cumulative survival was assessed on
the same days. Whole wet weight was measured with an electronic scale to
0.001 g. Also at day 360, oysters from all 3 groups were sampled for
genetic confirmation and assessment of gonadal development.

All statistical analyses were performed with Statistical Program
for Social Sciences (SPSS) 16.0, and significance for all analyses was
set at P < 0.05 unless noted otherwise. Shell height and wet weight
were transformed to logarithms to ensure normality and homoscedasticity.
Fertilization level and survival data were arcsine-transformed before
analysis. Differences in growth and survival among 3 groups (GG, HG, and
HH) were analyzed with l-way ANOVA, followed by multiple comparison
tests (LSD).

Genetic Confirmation

The identity of parents and selected progeny were confirmed using
the ITS2 (internal transcribed spacer 2) marker as described by Wang and
Guo (2008). DNA was extracted from ethanol-fixed samples using the
TIANamp Marine Animals DNA kit (Tiangen). Primer sequences for ITS2 were
5'-GGG TCGATGAAGAACGCAG (5.8S forward) and 5'-GCTC
TTCCCGCTTCACTCG (18S reverse). PCR was performed in a 25-[micro]L volume
containing 1.5 mM MgC12, 0.2 mM dNTP, 0.2 [micro]M of each primer, 20 ng
template DNA, 1 U Taq polymerase, 2.5 [micro]L 10 x PCR buffer, and 0.4
mg/mL BSA. The thermal cycler protocol consisted of an initial denature
at 95[degrees]C for 5 min, 30 cycles of 95[degrees]C for 1 min,
62.5[degrees]C for 1 rain, and 72[degrees]C for 1 min, with a final
extension at 72[degrees]C for 5 min. Three controls were included in
PCR- one with DNA from a known C. hongkongensis, one with DNA from a
known C. gigas, and the other with mixed DNA of the 2 species. Amplified
fragments were separated on 2% agarose gels containing 0.2 [micro]g/mL
ethidium bromide, and were visualized under a UV transilluminator
(BIORAD). Species are identified by fragment size difference (Wang &
Guo 2008).

PCR fragments were cloned and sequenced to confirm species identity
further. PCR products were purified using U-NIQ-5 Column PCR
Product's Purification Kit (San-gon, Shanghai), ligated into pMD
18-T vector following the instructions of the Takara DNA Ligation Kit
ver. 2 and were used to transform competent JMI09 Escherichia coli cells
using standard protocols. Recombinant colonies were identified by blue
white screen. Inserts of the expected size were detected via restriction
enzyme digestion (Eco RI and Hind III). Vector DNA containing the
desired inserts was purified further using the Pharmacia EasyPrep Kit,
and sequencing was performed in both directions on an ABI PRISM 377XL
DNA Sequencer using the ABI PRISM BigDyeTM Terminator Cycle Sequencing
Ready Reaction Kit with AmpliTaq DNA Polymerase FS (Perkin Elmer).

RESULTS

Fertilization and Embryonic Development

A notable difference between C. gigas and C. hongkongensis is the
size of their eggs. The eggs of C. gigas were larger than those of C.
hongkongensis. The eggs of C. gigas had an average diameter of 50.6
[micro]m, those of C. hongkongensis had a diameter of 40.6 [micro]m
(Table 1), and the difference is highly significant (P < 0.001).

Of 9 replicates of 2 x 2 crosses, 4 replicates were incomplete
because of poor gamete quality and problems during larval culture that
seriously affected intraspecific controls, and abandoned. Data from 5
complete replicates are presented here. Fertilization level in the 2
intraspecific crosses was high, ranging from 96.0-99.1% in C. gigas and
67.2-92.6% in C. hongkongensis. Eggs of C. hongkongensis could be
fertilized readily by C. gigas sperm in the HG cross, although the
fertilization level (61.6%) was lower than that in intraspecific crosses
(Table 1). On the other hand, no fertilization was observed in the
reciprocal cross, C. gigas eggs x C. hongkongensis sperm (GH cross),
despite adding 50% more sperm.

Embryonic development in the HG hybrid cross appeared normal, and
no apparent abnormalities were noticed. Embryos in all 3 crosses except
GH, which that had no fertilization, reached D-stage at 24-27 h
postfertilization. Survival of fertilized eggs to D-stage larvae in the
HG hybrid cross was 56.3%, significantly (P < 0.001) lower than in HH
(75.6%) or GG (97.1%: Table 1). Survival to D-stage of the 2
intraspecific crosses was also significantly (P < 0.01) different.

Survival and Growth

At day 1, or 24-27 h postfertilization, the height of D-larvae
differed significantly (P = 0.005) among the 3 groups: 62.8 [micro]m in
HH, 61.5 [micro]m in HG, and 70.9 [micro]m in GG, closely following
differences in egg size. From day 1, larvae in the HG hybrid cross were
always smaller than those of the 2 intraspecific crosses (Fig. 1 A).
Growth of HH larvae was slower than GG larvae during the first 10 days,
but no significant difference was observed from day 13-19.

Survival of D-larvae to different days postfertilization differed
significantly among groups and always followed the order of GG > HH
> HG (Fig. 1 B). Only 2.8% of D-larvae in the HG cross survived to
day 19, compared with 43.9% in the HH cross and 63.6% in the GG cross
(Table 1). HG hybrids also had lower metamorphosis success, with only
0.6% of D-larvae reaching spat stage at day 90 compared with 36.2% in HH
and 49.4% in GG. From day 90-360, survival of hybrid spat (78.9%) was
actually higher than that of HH (56.5%) and GG (64.1%) spat (Table 1).
During the same period, spat from the HG hybrid cross were consistently
smaller than those from the HH and GG crosses (Fig. 2). At day 360, HG
oysters were 62.6 mm in shell height and 64.1 g in whole weight compared
with 81.3 mm and 92.4 g for HH oysters, and 114.6 mm and 125.0 g for GG
oysters. The differences were highly significant (P < 0.001 ; Fig.
2).

Genetics Confirmation

At day 360, we sampled oysters from the 3 crosses for genetic
confirmation and to assess gonadal development. All parents used in the
5 replicates were unambiguously identified as C. gigas or C.
hongkongensis with the ITS2 marker (Fig. 3). Amplification in C. gigas
and C. hongkongensis produced single bands at about 800 bp and 720 bp,
respectively. All hybrid spat produced 2 bands, corresponding to the 2
parental species.

[FIGURE 1 OMITTED]

To confirm that the 2 bands are indeed from C. gigas and C.
hongkongensis, we sequenced fragments amplified from C. gigas, C.
hongkongensis, and the 2 fragments from hybrids. Sequences obtained from
the 800-bp fragment from C. gigas and hybrids both matched ITS2 of C.
gigas in GenBank AF280610.1 (e-value = 0.0, identities = 99%). There was
no ITS2 sequence for C. hongkongensis in public databases. From the
720-bp fragment amplified from C. hongkongensis, we obtained a 720-bp
sequence and deposited it in GenBank under accession no. GU338879. From
the 720-bp fragment amplified from hybrids, we obtained a 719-bp ITS2
sequence that matched the ITS2 of C. hongkongensis we previously
obtained: GU338879 (e-value 0.0, identities = 100%). Although there are
some minor variation in sequences between different oysters and clones
(because ITS2 has many copies per genome and can be variable), genetic
analysis indicates clearly that oysters from the HG hybrid crosses
contain ITS2 of both C. gigas and C. hongkongensis, and therefore are
true hybrids.

To assess the reproductive potential of HG hybrids, we examined
gonadal samples microscopically for the presence of gametes (mature eggs
and mobile sperm). All oysters from the GG and HH crosses were fully
mature and had either eggs or sperm (Fig. 4). Of the 153 hybrid oysters,
90% or 60.8% had no eggs or mobile sperm (respectively), 45 had mature
eggs, 12 had mobile sperm, and 3 were hermaphrodites (Table 2). Even
when hybrids contained mature gametes, most of them showed retarded
gonadal development in appearance (Fig. 4) and relatively few gametes
compared with oysters from intraspecific crosses.

Sperm-egg interaction involves gamete recognition proteins (GRPs),
and one of the GRPs is bindin, which is found in the acrosome of sperm
(Vacquier & Moy 1977, Vacquier 1998). Bindins are lectins that bind
specifically to receptors on the surface of eggs. Assuming all
Crassostrea species are evolved from a common ancestor, the fact that C.
hongkongensis and C. sikamea sperm cannot fertilize eggs of C. gigas
suggests that bindins of C. hongkongensis and C. sikamea or receptors on
eggs of C. gigas may have gone through significant changes. The former
scenario may be more plausible because C. sikamea sperm have also lost
their ability to fertilize eggs of C. ariakensis (Xu et al. 2009). It
has been shown that bindins of C. gigas are extremely diverse (Moy et
al. 2008). The high diversity of bindin may have given C. gigas sperm
the ability to fertilize eggs of several Crassostrea species, including
C. hongkongensis (this study), C. sikamea (Banks et al. 1994), and C.
virginica (Allen et al. 1993). Such interpretation is largely
speculative, because hybridization and bindin diversity have not been
studied extensively in oysters. Further studies are needed to elucidate
the role of GRPs in oyster speciation.

[FIGURE 3 OMITTED]

It is not surprising that hybrid larvae show slow growth and
reduced survival compared with larvae of parental species. The oysters
C. gigas and C. hongkongensis are believed to have diverged 28.8 million
y ago (Ren et al. 2010). Some genome incompatibility may have developed
during this long divergence, causing some hybrid dysfunction and
affecting the growth and viability of hybrid larvae. Hybrid crosses
between other Crassostrea species have also exhibited lower survival and
poor growth compared with intraspecific crosses (Allen et al. 1993, Xu
et al. 2009). Hybrid juveniles and adults were also smaller than
parental species in this study, which is also true for C. sikamea x C.
ariakensis hybrids (Xu et al. 2009). This is clearly different from the
usually positive heterosis observed in intraspecific hybrids (Cruz &
Ibarra 1997, Zheng et al. 2006, Hedgecock & Davis 2007).

The observation that most hybrid oysters did not produce mature
gametes at 1 y of age when all C. gigas and C. hongkongensis were fully
mature suggests that genome incompatibility between C. gigas and C.
hongkongensis also affected the reproductive potential of some hybrids.
On the other hand, some hybrids produced some functional gametes and may
be partly fertile. Fertile hybrids have been observed between C. gigas
and C. sikamea (Camara et al. 2008), and between C. gigas and C.
angulata (Huvet et al. 2002), although whether the latter 2 species are
different species has been a subject of debate (Menzel 1974, Huvet et
al. 2002). A recent study has classified them as 2 subspecies, C. gigas
gigas and C. gigas angulata, based on their level of divergence (Wang et
al. 2010). The finding of fertile hybrids opens up the possibility of
gene introgression between different species. Whether HG hybrids can be
backcrossed to their parental species and produce viable offspring
requires further investigation.

[FIGURE 4 OMITTED]

In conclusion, our study demonstrates that both prezygotic and
postzygotic barriers to hybridization exist between C. hongkongensis and
C. gigas, although none of them are complete. Fertilization is possible
in one direction but not in the other direction. Hybrids exhibit slow
growth, reduced survival, and retarded gonadal development, although
some can survive to sexual maturation and produce some functional
gametes. These fertile hybrids may be valuable in gene introgression
between C. gigas and C. hongkongensis, both of which are major
aquaculture species. On a theoretical note, these findings raise the
issue of whether the level of hybridization barrier observed is
sufficient to maintain species integrity should the 2 species become
sympatric again.

ACKNOWLEDGMENTS

We thank Xirui Guo, Qiang Ma, and Junwei Sun of Laodong Aquaculture
Breeding Company for operational support; Fei Xu for providing oyster
broodstock; Xue Yi and Chenchen Zhang for their help with larval
rearing; and Jiaqi Su, Hui Zhang, Huanqiang Sun, Liqiang Zhao, Xin Sun,
Shaowen Li, and Yan Wang for assistance in the hatchery and grow-out. We
thank Haiyan Wang, Jinhai Wang, Zhifei Yu, Changwei Shao, and Feng Gao
for their kind assistance with molecular identification. This research
was supported by grants from the National Natural Science Foundation of
China (31172403) and the National Basic Research Program of China
(2010CB 126406).